Abstract Rabies virus (RABV) and related pathogens of the lyssavirus genus cause a fatal neurological disease that leads to at least 55,000 deaths annually. Whereas the current rabies vaccine is save and efficacious, it protects only against infection by phylogroup I lyssaviruses but is ineffective against emerging phylogroup II pathogens that also cause rabies. This efficacy gap equally extends to the human rabies immunoglobulin, which is used in conjunction with vaccination for post-exposure prophylaxis against rabies. Independent of the phylogroup type of the causing agent, no reliable treatment option is currently available for rabies after the onset of clinical symptoms. The development of a novel drug discovery platform for the identification of next- generation anti-lyssavirus drug candidates with activity against all clinically relevant lyssavirus phylogroups is therefore urgently needed. We hypothesize that shelf-stable and cost-effective small molecule inhibitors with broad anti-lyssavirus indication spectrum will improve global access to life-saving anti-rabies post-exposure prophylaxis, prepare against emerging lyssavirus pathogens of different phylogroups, and establish a foundation to explore innovative treatment options for symptomatic rabies. Building on our highly complementary multiple-year expertise in drug discovery assay development, automated drug screening, lyssavirus biology, and RABV pathogenesis, we have formed an interdisciplinary team to ultimately address this unmet clinical need. In pilot studies, we have generated a first-in-class RABV reporter strain harboring nano-luciferase in place of the G protein encoding open reading frame (RABV-?G-nanoLuc) that induces a single-cycle infection and can be maintained under standard biosafety conditions, developed a novel anti-RABV drug screening protocol suitable for automation, and miniaturized the assay to 384-well scale. In parallel, we have established direct and orthogonal counterscreens for hit candidate confirmation and lyssavirus indication spectrum assessment, and have developed a comprehensive panel of assays for early-stage mechanistic characterization. Building on this tangible foundation, we will in this 3-year pre-therapeutic discovery project fully validate the high-throughput screening (HTS) protocol and apply the assay to a drug discovery campaign using the HTS facilities established in our laboratory (specific aim 1). Emerging hit candidates will be verified in counterscreens, toxicity and indication spectrum determined on established cell lines and primary cells of human and murine origin, and the potential for synthetic optimization assessed (specific aim 2). To short-list a subset of structurally and mechanistically distinct lead candidates that warrant full synthetic development, confirmed hits will be mechanistically characterized, viral escape profiles determined, and the potential for brain penetration evaluated (specific aim 3).